Neurons within the developing nervous system interconnect with incredible precision and specificity. Neuronal processes are guided to their correct targets by cues in the environment that either attract them onto appropriate pathways or discourage them from extending on inappropriate pathways. Several families of signaling molecules that act as guidance cues have been identified over the past decade. Typically, these cues have been identified using genetic screens, through the pairing of in vitro bioassays with biochemical purifications, or by the characterization of selected candidate molecules. Although each approach has powerful advantages, none of them constitute a rapid, systematic, high throughput screen for guidance activities. The goal of this proposal is to develop a practical in vitro expression screen for axonal repellents. The aim would be to screen a large library of recombinant proteins in a relatively short period of time using a bioassay for repellent activity. By identifying active molecules through a positive selection criteria, this approach will complement more traditional forward genetic screens that rely on loss of function phenotypes to detect new activities. Candidate cues discovered in the screen could subsequently be tested for guidance activity by reverse genetic and knockdown approaches. The discovery of novel axonal guidance cues will advance our understanding of the mechanisms underlying the formation and plasticity of neuronal interconnections, and may ultimately help us improve axon regeneration in the damaged nervous system.